The enormous size of microbial populations continues to be a great asset in a variety of genetic studies including searches for valuable mutant microorganisms where it is possible to effectively select a rare gene type or mutant microorganism. Mutant cell varieties of a single strain of microorganism (procaryotic, eucaryotic or viral) have classically been isolated by a variety of methods including positive cell "selection" and differential "screening" of individual cell colonies.
Selection is used to isolate mutant varieties of microorganisms when a genetic alteration provides the microorganism with a positive growth advantage over its parental population. For example, acquisition of antibiotic resistance can be used to select such mutants on a nutrient agar surface containing the antibiotic. Another example is the acquisition of a biosynthetic gene enabling the organism to grow in a culture medium that would not otherwise support growth.
Mutants which cause only a small change, a negative change (decrease) or no change in the rate of cell growth are identified by screening of cell colonies. The colonies are produced by multiplication of a single cell. Screening is used to detect mutant microorganism species producing beneficial increases (or decreases) in the synthesis (or the breakdown) of important primary and secondary metabolites for example. However, screening to identify a mutant colony can require examination of tens of thousands of individual colonies to determine the presence of mutants. Replica plating is one screening technique. Thereafter, tedious visual comparison of petri dish pairs are required as well as relatively large amounts of selective and/or restrictive materials which serve to differentiate the mutant from its parent. In general, although screening techniques, are highly effective in achieving the desired result, they are labor and material-intensive requiring examination of many individual colonies, usually in petri dishes.
Recently, a technology has emerged which provides for encapsulating biological material such as living tissue, individual cells, viruses, and biological macromolecules within a semi-permeable membrane. The basic approach in this technique involves suspending the biological material to be encapsulated in a physiologically compatible medium containing a water soluble substance that can be made insoluble in water, e.g., a gel, to provide a temporary environment for the biological material. The medium is formed into droplets containing the tissue and gelled by changing any one of a variety of ambient conditions. These temporary capsules are then subjected to a treatment which results in the production of membranes with a desired permeability (including impermeable membranes). One such technique, is exemplified in U.S. Pat. No. 4,352,883 entitled "Encapsulation of Biological Material", the disclosure of which is incorporated herein by reference.
A description of a technique for separating cells having desired properties from a large population is found in U.S. Pat. No. 4,401,755 entitled "Process for Measuring Microbiologically Active Material" which discloses a method for measuring an unknown quantity of microbiologically active material utilizing unencapsulated microdroplet techniques similar to the first stage process of U.S. Pat. No. 4,352,833. The disclosure of U.S. Pat. No. 4,401,755 is also incorporated herein by reference. After preparing a suspension of gel microdroplets, the suspension is processed in an apparatus having the capability of sensing a physical characteristic of individual gel microdroplets to determine the presence or absence of a desired physical characteristic of the biological material in such a droplet.
It is apparent that a need to develop new mutant isolation techniques exists which will reduce the costs and time spent in selection and screening processes used to isolate mutants from their respective parent populations. Microencapsulation technology, as described in the above referenced patents, provides the potential for solving a variety of problems including the labor and cost excesses of prior art mutant microorganism isolation techniques.